Malignant tumors severely threat the human life. At present, the conventional means for the treatment of malignant tumors are surgery, radiotherapy and chemotherapy, which, however, cannot achieve satisfactory effect on most tumors. The therapeutic index of most chemotherapeutics is very low, that is, their therapeutic dose and toxic dose are almost the same. Thus the aforementioned therapeutic means are accompanied with substantive toxic effect, including the life-threatening suppression of bone marrow. Therefore, it is very important for tumor treatment to develop methods which selectively kill the tumor cells but not affect the normal cells. These methods mainly rely on the characteristic marker of tumor cells, and their therapeutic effects depend on whether said tumor marker is strictly restricted to tumor cells.
Telomere is a structure of eukaryotic cells, which protects the end of chromosomes. The telomere of normal cells shortens by 50-200 nucleotides after each cell division. When the length of the telomere decreases to a certain degree, the cells will die. While in malignant cells, the telomerase is activated, whereby elongating the shortened telomere and maintaining the stability of the chromosome. Then the cells escape from death and become immortal. It means that the tumor cells can propagate continuously. Therefore, the activation of telomerase plays an essential role in the onset and development of tumor. The telomerase activity is positive in about 90% tumor cells, while it is negative in most of the normal cells. Based on this fact, the telomerase can be used as a characteristic marker of tumor cells.
At present, the telomerase in human is known to be consisted of the following three components: (1) RNA component (hTERC), which acts as an endogenous template for repeated synthesis of telomere; (2) telomerase binding protein (TEP1); and (3) telomerase catalytic subunit (abbreviated as hTERT), also named as telomerase reverse transcriptase. RNA component and hTERT are essential for the activity of telomerase. Recent study further shows that hTERT plays a critical role in the telomerase activity, and is highly expressed in most tumor cells or tumor cell lines, while is expresses at a lower level or even not expressed in normal cells. Thus it is believed that the promoter of hTERT is in an activated state in most tumor cells.
Various researches have been carried out on tumor therapy, which aims at using telomerase as the target. These studies include the following: (1) tumor therapy, which aims to inhibit telomerase activity; (2) gene therapy, which uses telomerase promoter (i.e. hTERT promoter and/or telomerase RNA component promoter) to drive an apoptosis gene or suicide gene; and (3) virotherapy, which employs a tumor-specific propagating virus controlled by the hTERT promoter.
However, the aforementioned therapy still has significant defects. Firstly, the telomerase activity is negative in about 10% of the human tumors. In these tumor cells, the length of the telomere is maintained by the mechanism termed as Alternative Lengthening of Telomere (ALT). Furthermore, due to the heterogenicity of tumor cells, the fact that the telomerase activity is positive in certain tumor cells of a patient does not means that the telomerase activity is positive in all of the tumor cells in said patient. ALT mechanism may also exist in said patient. Thus antitumor strategy only aiming at the telomerase cannot kill all the tumor cells. Studies have shown that, telomerase inhibitor can inhibit the telomerase activity, but also enhance ALT mechanism in tumor cells, whereby not being efficient to kill the tumor cells. Secondly, in human body, the telomerase activity is also positive in germ celler, hemopoietic stem cells and diverticulum cells in the gastrointestinal tract. Thus the tumor therapy aiming at the telomerase may cause toxicity to said cells.
In the last decade, gene therapy was developed in clinical research. Clinical practice shows that, gene therapy is very safe, but its efficiency in treating tumor is also very low, and in some cases, it even has no therapeutic effect. Although there are various reasons for said defect, the main reasons are lower efficiency of in vivo transfection of the vector system, lower expression of the cancer therapeutic gene and being unable to target tumor cells. Thus the cancer therapeutic gene cannot be transfected to sufficient tumor cells and be highly expressed in said cells, which adversely affects the therapeutic effect of gene therapy in tumor clinical practice. It is a key point in tumor gene therapy that how to increase the transfection efficiency of the vector system, how to increase the expression of the cancer therapeutic gene and the specificity to tumor cells.
In recent years, due to the rapid development in virology, molecular biology and oncology, the genomes of various virus have been sequenced, and their gene structures and functions have been studied in detail, thus virus genes can be effectively modified. The modified virus has enhanced ability to transfect tumor cells, replicate in cells as well as increased lysis effect. Whereas, in normal cells, said ability is decreased or even lost. Thus such virus can replicate selectively in tumor cells, and propagate to thousands or even millions folds, causing tumor cell lysis. Upon cell lysis, new virus is released, and again transfects and propagates in tumor cells, until all the tumor cells are killed. Since such virus cannot propagate in normal cells, it has little effect on them. Such virus, which specifically propagates in tumor cells, is termed as tumor-specific propagating virus. As yet, about ten types of tumor-specific propagating virus are in clinical test.
However, virotherapy, which only employs a tumor-specific replicating virus, has its own limits. Firstly, due to the complicated mechanism of tumor formation, and The heterogenicity of tumors, there is obvious difference among patients, tumors or even cells in a tumor,
With regard to the telomerase, the fact that the telomerase activity is positive in certain tumor cells of a patient does not mean that the telomerase is positive in all the tumor cells of said patient. Thus the virus employing a certain tumor mechanism to propagate cannot kill all the tumor cells. Secondly, the diffusion of virus may be inhibited by various factors in tumors, such as fibrosis, existence of normal cells and necrosis region. Thirdly, in certain tumors, the insufficient expression of the receptor of the virus (e.g. Coxsackie virus receptor) inhibits the infection of said virus. Fourthly, the immune response of the patient to the viruses also inhibits the proliferation and diffusion of virus.
ONYX Pharmaceutical Company (USA) applied only E1b 55 kDa protein deleted virus (ONYX-015) to treat tumor, but only achieved 15-20% efficiency in clinics (Nemunaitis, J. et al, Cancer Res., 2000, 60(22):6359; U.S. Pat. No. 5,677,178; and U.S. Pat. No. 5,801,029).
Therefore, more effective and specific tumor therapy with minimized side-effect is urgently desired.